We are studying signaling networks in the retinal pigment epithelium (RPE) with special emphasis on differentiation/dedifferentiation pathways and protection against oxidative or inflammatory stress. The role of signaling pathways in RPE differentiation and de-differentiation is an important focus of our research. Divergence from or convergence to the phenotype of native RPE is a common theme of much RPE cell culture research and this has an important impact on the potential use of RPE cells in cell therapy for retinal degenerations. In particular, given the likely importance of noncoding RNAs (including microRNAs (miRNAs) and long noncoding RNAs (lncRNAs)) as regulators of gene expression in the response of RPE cells to various signals, we are interested in determining how changes in miRNAs and lncRNAs expression in RPE cells affect differentiation/dedifferentiation, and how their expression may be altered with manipulation of RPE cells. In the past year we have made progress in the following areas: 1) We continued a project focusing on lncRNA discovery in RPE, to identify and characterize lncRNAs important in RPE gene regulation. Non-coding RNAs play an important role in regulating the expression of genes involved in numerous biological processes including cellular differentiation. Most ncRNA studies have focused on miRNAs, as we have previously studied in RPE, while few studies have investigated how lncRNAs, a group of RNAs (>200 nucleotides, generally with no protein coding potential) regulate gene expression and cellular differentiation in RPE. To regulate RPE-specific functions we expect there to be a set of highly RPE-specific lncRNAs. Also, there is likely to be an important role of lncRNAs in the differentiation of RPE cells. As native human RPE is difficult to obtain, we are using bovine RPE to develop the paradigm, working with native and RPE primary cultures to identify lncRNAs differentially expressed between RPE and retina, and between native bovine and cultured RPE cells. Currently, several lncRNAs (TUG1, MALAT1, MEG3 and others) have been discovered to regulate normal visual function and may potentially contribute to dysfunction of the retina. We decided to extend these analyses of lncRNA genes to the retinal pigment epithelium (RPE) to determine whether there is conservation of RPE-expressed lncRNA between human and bovine genomes. We did a reconstruction of bovine RPE lncRNAs based on genome guided assembly. Next, we predicted homologous human transcripts based on whole genome alignment. We found a small set of conserved lncRNAs that could be involved in signature RPE functions that are conserved across mammals. However, the fraction of conserved lncRNAs in the overall pool of lncRNA found in RPE appeared to be very small, perhaps reflecting a fast and flexible adaptation of the mammalian eye to various environmental conditions. A manuscript describing these results was published in this reporting period. 2) We continued a project studying stable intronic sequences and exon skipping events in the human RPE65 gene. Currently, there is much interest in intronic sequence-containing long non-coding RNAs and the role of intronic transcription in regulation of cellular metabolism and fate. Several stable intronic sequence RNAs (sisRNAs) were recently implicated in regulation of parental genes. To investigate transcription from introns of the RPE65 gene, we analyzed RNA-seq and Nanopore sequencing data from different cell models of human retinal pigment epithelium (RPE) and native bovine RPE. We discovered putative stable poly-adenylated transcripts with sequences corresponding to intronic regions of the RPE65 gene in the cytoplasm of RPE cells. These stable intronic sequences could be important for RPE65 transcription, splicing or translation. We also analyzed alternative splicing events in RPE65. Frequent exon skipping events involving exons 2, 3, and 7 were detected. The rate of these events was much higher in human RPE cell cultures compared with native RPE suggesting potential problems in RPE65 translation mRNA in cell cultures. A manuscript describing these results was published in this reporting period. 3) We continued a project to study the role of the lncRNA LINC00276 in RPE. RNASeq analysis provided a comprehensive view of differentially expressed lncRNAs in differentiated 4-month old ARPE-19 cells relative to 4-day old cells. From these data, we observed a number of lncRNAs that were differentially regulated with fold change of 2.5 in differentiated ARPE-19 cells and which show a differential expression pattern between 4-da and 4-mo cultured cells. The expression of one of these in particular, LINC00276, was increased >200-fold in 4-mo cells compared to 4-da cells. Knockdown of LINC00276 negatively affected expression of various RPE-preferential transcripts, while its overexpression enhanced expression. By silencing LINC00276, we observed a decrease in the expression of genes associated with RPE differentiation such as, MITF, TRPM1, TRPM3 and miR-204/211, while LINC00276 overexpression increased their expression. Silencing LINC00276 also decreased RPE-characteristic genes such as RPE65, TYR and MERTK, while altering the expression of genes involved in Wnt signaling pathway. We have determined that LINC00276 is preferentially expressed in native human RPE. These studies are ongoing. 4) We continued a project to examine the expression of secreted proteins (secretome) in differentiated ARPE-19 cultured in DMEM with pyruvate for 4 months and exhibiting native-like RPE phenotype. We are also investigating secretion of exosomes from ARPE-19 as a function of their differentiation to determine proteins and/or RNAs secreted. We continue to collaborate with sections in the LRCMB and with other laboratories and sections (Molecular Structure and Functional Genomics, Laboratory of Immunology), as well as with extramural labs in the analysis (HPLC and mass spectrometry) of retinoids, lipids, and other compounds.